Saturday, May 4, 2024
5/4/2024
PROJECT SUMMARY/ABSTRACT A wealth of new tools can directly control output of specific neurons on fast (e.g., optogenetic) or sustained (e.g., chemogenetic) time scales. In contrast, almost no methods exist for selectively modulating communication between defined cells at the synaptic level, which is key to understanding how functional connectivity creates percepts, engrams and actions. Here, we advance a novel strategy for selectively modulating synaptic transmission, Interluminescence. This approach uses bioluminescent light from a presynaptic axon terminal, generated by a luciferase, to modulate an opsin in its postsynaptic target under experimenter-controlled introduction of a small molecule (luciferin). A challenge in developing Interluminescence is generating sufficient photon density across the synapse. To address this challenge, we developed two separate methods that target the luciferase to the synaptic cleft. These two Interluminescent methods offer distinct features for experimenter needs. To provide sustained and synapse-specific regulation, the ‘Persist-Int’ strategy places a luciferase in the synaptic cleft tethered to the presynaptic terminal, and an opsin in the opposing postsynaptic membrane. In this configuration, light generation creates sustained and activity-independent modulation. In the complementary ‘Act-Int’ strategy, luciferase is released into the synaptic cleft in response to presynaptic activity, a synapse-specific form of activity-dependent modulation. In addition to their distinctive features, these Interluminescence methods are unique in providing synapse-specific pre- to post-synaptic regulation under experimenter control. Two further steps are crucial to deliver a reliable toolset that can be readily adopted, making Interluminescence useful to a broad community. First, we need to characterize in detail the impact of Interluminescence in individual neurons. To this end we will conduct experiments in cell culture and brain slices, recording from individual postsynaptic neurons and endogenous brain circuits in vitro (Aim IA-B). Second, we need to examine the impact of Interluminescence in vivo. Here we will test Interluminescence in anesthetized and awake animals, in the well-characterized barrel cortex (Aim IC). In two additional Aims, we will begin to elaborate this platform technology by testing a novel activity-dependent luciferase regulation mechanism (Aim II), and by engineering light emitting components to further increase the temporal and spatial resolution of Interluminescence (Aim III). The validated technology will enable pursuing a new class of research questions, both basic and translational, that currently cannot be addressed with available technology.
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